The goals of this proposal are to examine how the fetal neuroepithelial stem cells support both neurogenesis and ependymogenesis, and to determine how ventricular enlargement, or hydrocephalus, affects ependymogenesis and associated stem cell functions. Ependymal cells are arranged as a monolayer along the ventricle walls and act as the functional units of transport between the cerebrospinal fluid (CSF) and interstitial fluid (ISF). A healthy ependyma circulates CSF and facilitates the clearance of toxins from the ISF of the brain parenchyma. During mid-gestation, sufficient numbers of ependymal cells are generated from neuroepithelial stem cells to cover the entire ventricle surface, but in hydrocephalus, the periventricular stem cell niche is called upon to generate many more ependymal cells to cover the expanding ventricles. We will test the hypothesis that ventricle expansion exhausts the stem cell pool and results in insufficient coverage of the ventricle surface, periventricular gliosis, and loss of critical transependymal transport/clearance functions. Experiments are designed to document the dynamic supply and demand relationship between stem cells and their progeny ependymal cells, in both human tissue and mouse models of hydrocephalus. In Aim 1, we will create spatiotemporal maps of ependymal cell generation and determine the relationship between the distribution of ependymal cells and stem cell numbers/organization in normal fetal and postnatal human periventricular brain tissue. Similar studies will be performed in mouse, supplemented with time-lapse stem cell lineage tracing using the piggyBac in utero electroporation system, to track stem cell fates and test whether ependymogenesis results in stem cell exit from the cell cycle. The resulting 3D spatiotemporal maps of ependymogenesis will serve as tools for evaluating abnormal ependyma development and stem cell niche changes in hydrocephalus. In Aim 2, we will examine several models of hydrocephalus to determine how the stem cell niche and ependymal cell coverage of the ventricle surface are affected. Cell organization, cell-cell junction complexes, alterations in transependymal flow and clearance mechanisms at the ventricle surface and stem cell dynamics will be elucidated. Data generated will provide insight into the regenerative potential of neuroepithelial stem cells for repair of the ventricle surface and how deficits in stem cell functions may contribute to the multiple neurologic pathologies associated with hydrocephalus.